key: cord-0805752-axvjfsrg
authors: Huang, Xianlong; Zheng, Zhiwen; Wu, Yixin; Gao, Meng; Su, Zhengding; Huang, Yongqi
title: 14-3-3 Proteins are Potential Regulators of Liquid–Liquid Phase Separation
date: 2022-02-10
journal: Cell Biochem Biophys
DOI: 10.1007/s12013-022-01067-3
sha: c382ed326c2f6cf1ec76118c51c8c7be57fb2755
doc_id: 805752
cord_uid: axvjfsrg

The 14-3-3 family proteins are vital scaffold proteins that ubiquitously expressed in various tissues. They interact with numerous protein targets and mediate many cellular signaling pathways. The 14-3-3 binding motifs are often embedded in intrinsically disordered regions which are closely associated with liquid–liquid phase separation (LLPS). In the past ten years, LLPS has been observed for a variety of proteins and biological processes, indicating that LLPS plays a fundamental role in the formation of membraneless organelles and cellular condensates. While extensive investigations have been performed on 14-3-3 proteins, its involvement in LLPS is overlooked. To date, 14-3-3 proteins have not been reported to undergo LLPS alone or regulate LLPS of their binding partners. To reveal the potential involvement of 14-3-3 proteins in LLPS, in this review, we summarized the LLPS propensity of 14-3-3 binding partners and found that about one half of them may undergo LLPS spontaneously. We further analyzed the phase separation behavior of representative 14-3-3 binders and discussed how 14-3-3 proteins may be involved. By modulating the conformation and valence of interactions and recruiting other molecules, we speculate that 14-3-3 proteins can efficiently regulate the functions of their targets in the context of LLPS. Considering the critical roles of 14-3-3 proteins, there is an urgent need for investigating the involvement of 14-3-3 proteins in the phase separation process of their targets and the underling mechanisms.

The 14-3-3 proteins are ubiquitously expressed in various tissues and mediate many cellular signaling pathways. The name "14-3-3" was given because these proteins elute in the 14th fraction of bovine brain homogenate on the DEAE cellulose column and the 3.3 fraction in the subsequent starch gel electrophoresis [1] . They interact with numerous protein targets, including kinases, phosphatases, transmembrane receptors, and transcription factors [2, 3] . Through these protein-protein interactions, 14-3-3 proteins participate in cell cycle regulation, gene expression control, apoptosis, signal transduction, and many other vital biological processes [4] [5] [6] . Dysregulation of 14-3-3 protein expression has been observed in several cancers [7] . Furthermore, 14-3-3 proteins have chaperone activity which may play a role in neurodegenerative disease progression [8] . Since 14-3-3 proteins are potential therapeutic targets, many 14-3-3 modulators have been discovered/designed to target these specific interactions [9, 10] .

The binding targets of 14-3-3 proteins usually carry specific motifs containing phosphorylated serine or threonine. Most of these motifs are embedded in intrinsically disordered regions (IDRs) [11, 12] . In contrast to traditional folded protein domains, IDRs do not fold into stable three dimensional structures in the free state. Their amino acid compositions significantly differ from those of folded proteins. Generally, IDRs lack bulky hydrophobic amino acids and are enriched in charged amino acids [13, 14] . While IDRs are unfolded, they perform critical functions. Structural disorder facilitates post-translational modifications (PTMs), association/dissociation kinetics, conformational changes, and so on [15, 16] . Consequently, numerous IDRs are involved in cellular regulation and signal transduction [17] [18] [19] .

Recently, IDRs have become a theme of many research efforts because they are closely associated with liquid-liquid phase separation (LLPS), which underlies the formation of membraneless organelles and cellular condensates [20] [21] [22] [23] [24] [25] . Increasing evidences suggest that many types of biomolecular condensates, such as stress granules, P granules, and nuclear speckles, are formed through LLPS [24, [26] [27] [28] [29] . Enriching interacting components inside droplets has been shown to promote protein aggregation [30] [31] [32] , enzyme reaction [33, 34] , gene expression [35] [36] [37] , and virus replication [38, 39] . LLPS is driven by multivalent interactions and can be readily regulated by effector molecules or PTMs [22, 29, 40] .

To date, 14-3-3 proteins have not been reported to undergo LLPS alone or regulate LLPS of their binding partners. However, the high LLPS propensity of 14-3-3 binding partners and the bivalent nature of 14-3-3 proteins suggest that 14-3-3 proteins could be potentially involved in LLPS regulation. In this review, we first summarized the interacting modes of 14-3-3 and mechanism of LLPS, and then we proposed that 14-3-3 proteins could be recruited into the droplets and regulate the phase separation behavior of their binding partners.

The 14-3-3 proteins are a family of highly conserved scaffold proteins. They are expressed in various types of tissues [41] [42] [43] [44] [45] . In mammals, seven 14-3-3 isoforms encoded by different genes have been identified: α/β, γ, ε, δ/ζ, η, θ, and σ, where α and δ are the phosphorylated forms of 14-3-3β and ζ, respectively [46] [47] [48] . The length of human 14-3-3 isoform varies from 245 residues to 255 residues. Each 14-3-3 protein sequence contains a divergent N terminus, a divergent C terminus, and a conserved core region. A phylogenetic tree generated from sequence alignment is shown in Fig. 1a . 14-3-3 proteins form stable homodimers or heterodimers with a cup-shaped structure (Fig. 1b) . Each protomer consists of nine antiparallel α-helices where α1 and α2 of one protomer interact with α3 and α4 of the other protomer to form the dimer interface [49, 50].

14-3-3 proteins interact with their binding partners primarily through the amphipathic binding grooves constituted by α-helices α3, α5, α7, and α9 (Fig. 1b) . Three distinct 14-3-3 binding motifs are identified: motif I [RXXp(S/T)XP/ G], motif II [RX(F/Y)Xp(S/T)X(P/G)], and motif III [p(S/T) X 1-2 -COOH], where p(S/T) is a phosphorylated serine or threonine, and X is any type of residue [51-55]. The three conserved basic residues Lys49, Arg56, and Arg127 within the amphipathic groove form ionic and hydrogen bonds with the phosphorylated residue ( Fig. 1c) [49]. Although the sequences of the seven 14-3-3 isoforms are highly conserved, they bind to the same target with different affinities. Interestingly, their binding affinities obey a hierarchized profile with conserved relative K D ratios [56] .

It should be noted that not all interactions with 14-3-3 proteins require the above-mentioned phosphorylated motifs. Some non-phosphorylated peptides also bind to 14-3-3 proteins with high affinity [57, 58] . If a protein contains multiple motifs, they could occupy both amphipathic grooves within a 14-3-3 dimer simultaneously [59-62]. Besides the amphipathic grooves, the last two C-terminal αhelices are also shown to contribute to interactions with some partners [63] [64] [65] .

So far, hundreds of 14-3-3 binding proteins have been identified [66] [67] [68] . Affinity-purification/mass spectrometry combined with domain-based cluster analysis suggested that 14-3-3 proteins can potentially engage around 0.6% of the human proteome [69] . Overall, the 14-3-3 binding proteins show great structural and functional diversity. A recent Gene Ontology analysis of 14-3-3 partners showed that 25% are non-receptor Ser/Thr protein kinases, 20% are DNA binding proteins, 12% are cell cycle proteins, 10% are RNA-binding proteins, and several other categories [12] . Therefore, 14-3-3 binding proteins are extensively involved in cell cycle regulation, apoptosis, autophagy, signal transduction, and gene expression control [4] [5] [6] .

14-3-3 proteins regulate the function of their targets through several modes [70] : (i) A 14-3-3 dimer can act as a scaffold to bring two interacting proteins in close proximity, thus mediating the formation of protein complexes. For example, 14-3-3β mediates the interaction between Bcr and Raf [71] and 14-3-3ζ stabilizes interaction between α4 integrin and paxillin [72] . (ii) 14-3-3 proteins can occlude interactions between the target proteins and their effectors. Interaction between BAD and Bcl-2 promotes apoptosis. Datta et al. found that phosphorylation of BAD at Ser136 and Ser112 recruits 14-3-3 proteins, which disrupts the binding of BAD to Bcl-2 and thereby promotes cell survival [73] . (iii) 14-3-3 proteins can induce conformational changes on the target proteins, altering their activity. A wellstudied example is the serotonin N-acetyltransferase, an enzyme that controls the daily rhythm in melatonin synthesis. 14-3-3 proteins increase the activity of serotonin N-acetyltransferase by modulating the structure of the substrate-binding sites [74, 75] . Sometimes, different modes may work synergetically to modulate the activity of the 14-3-3 binding partners. For example, the binding site of 14-3-3ζ on the kinase domain of ASK1 is in close proximity to the kinase active site. Therefore, 14-3-3ζ may inhibit the activity of ASK1 by blocking its accessibility and/or affecting its conformation [76] .

By participating in numerous signaling pathways, dysregulation of 14-3-3 proteins is involved in many diseases [7, 8] . Expression levels of the majority of 14-3-3 isoforms are elevated in many tumors, such as meningioma [77] , astrocytoma [78] , glioblastoma [79] , lung cancer [80, 81] , breast cancer [82, 83] , and hepatocellular carcinoma [84, 85] . Because 14-3-3σ is a tumor suppressor, its expression in tumors is negatively regulated [77, 86] . By interacting with GSK-3β, LRRK2, or FOXO3a, or colocalizing with α-synuclein in the Lewy bodies or tau in the neurofibrillary tangles, 14-3-3 proteins are associated with Alzheimer's disease and Parkinson's disease [87] [88] [89] [90] [91] [92] [93] . Furthermore, 14-3-3 proteins are also associated with other neurological disorders, such as lissencephaly, schizophrenia, bipolar disorder, Williams syndrome, and Down syndrome [94, 95] .

Many proteins are able to undergo LLPS when the concentration exceeds a threshold value, in particular under crowded conditions. LLPS usually results in formation of liquid droplets in which proteins are enriched. The protein concentration inside the droplets can be tens of folds higher than that of the surrounding dilute phase [96, 97] . The phase separation behavior of a protein can be readily affected by several environmental conditions, such as salt concentration, pH, and temperature [98, 99] .

LLPS can be driven by multivalent interactions [22, 29] . Tandem repeats of binding element are multivalent, which is well examplified by the nephrin-NCK-N-WASP system [100] . Multivalence can also be achieved by oligomerization, as in NPM1 or SynGAP [101, 102] . Furthermore, IDRs form dynamic, multivalent interactions, such as cation-π Fig. 1 Sequence and structure analysis of 14-3-3 proteins. a A phylogenetic tree generated from sequence alignment of seven human 14-3-3 isoforms. b Two perpendicular views of 14-3-3σ dimer. Each protomer is shown as cartoon in rainbow color. The nine antiparallel αhelices in one protomer is labeled from α1 to α9. c Interactions between the pSer214 residue of tau peptide and the conserved basic residues Lys49, Arg56, and Arg127 within the amphipathic groove of 14-3-3σ. The tau peptide is shown as an orange tube, where the pSer214 residue is shown as red sticks. Residues Lys49, Arg56, and Arg127 of 14-3-3σ are shown as blue sticks. Crystal structure of the 14-3-3σ/tau complex (Protein Data Bank ID 4FL5 [60]) is used in b and c for illustration interactions, electrostatic interactions, and hydrophobic interactions [103] [104] [105] . The interaction mode between multivalent molecules can be described by the stickers-andspacers model [22, 106] , in which stickers are groups of residues forming attractive interactions, while spacers connect the stickers and influence their interactions.

As indicated by the stickers-and-spacers model, LLPS will be altered if the strength of interaction, the valence of molecules, the distribution of stickers, or the length of spacers is changed. PTM is a universal way to regulate LLPS [22, 40, 107, 108] . PTM can induce conformational changes or alter electrostatic interactions and hydrophobic interactions, thereby promoting or suppressing LLPS. Phosphorylation [109] [110] [111] [112] [113] , methylation [98, 111, [114] [115] [116] , acetylation [117, 118] , polyubiquitination [119, 120] , and PARylation [121, 122] , have been shown to alter LLPS of many proteins.

Proteins within a droplet can be generally divided into two classes [123] . The first class is scaffolds or drivers, which are multivalent and essential for LLPS. The second class is clients or regulators. Clients and regulators are dispensable for LLPS and are recruited into the droplets via interactions with the scaffolds or drivers. In promyelocytic leukemia (PML) nuclear bodies, the PML protein represents the scaffold, while SUMO-1, Daxx, and Sp100 are the clients and dynamically exchanged at the PML nuclear bodies [124, 125] . It has been shown that partition of clients into droplets varies with the scaffolds concentration or valency [123] . Recently, Orti et al. systematically analyzed the features of drivers, clients, and regulators, and observed some difference among them in disorder content, sequence composition, and prevalence of PTMs [126] .

Accumulated evidences suggest that LLPS underlies the formation of various membraneless compartments, such as stress granules, P granules, nuclear speckles, and nucleoli [24, 26-29]. Thus, LLPS plays critical roles in many cellular processes, including assembly of mitotic spindle [127] , cellular stress response [128, 129] , gene expression [35] [36] [37] , and RNA splicing [130] . Furthermore, LLPS is also associated with pathological processes, such as protein aggregation and virus replication [30-32, 38, 39]. Consequently, misregulation of LLPS may lead to the occurrence of diseases [31, 97, 131-133].

As discussed above, 14-3-3 proteins participate in numerous signaling pathways and regulate various biological processes, where LLPS may occur. However, the involvement of 14-3-3 proteins in LLPS has not been investigated. Since LLPS of 14-3-3 proteins has not been reported, we first predicted their LLPS propensities using the FuzDrop server [134] . FuzDrop prediction gives the probability of LLPS (p LLPS ) for human 14-3-3β, γ, ε, ζ, η, θ, and σ are 0.2884, 0.1581, 0.1586, 0.2169, 0.1364, 0.1318, and 0.2124, respectively, suggesting that all seven 14-3-3 isoforms will not phase separate spontaneously. We further analyzed the LLPS propensities for the binding partners of human 14-3-3 proteins. 1370 14-3-3 binding partners were collected from the IntAct database [135] and then subjected to FuzDrop prediction (Table S1) . Surprisingly, more than one half of the 14-3-3 binders show p LLPS values larger than 0.6 ( Fig. 2a) , suggesting that a large number of the 14-3-3 binders could undergo phase separation. A search of literature showed that LLPS of some 14-3-3 binding partners has been characterized experimentally ( Table 1 ). The Fuz-Drop prediction profiles of five representative 14-3-3 binders were shown in Fig. 2b . Given the high LLPS propensities of 14-3-3 binding partners and the above discussion on the modes through which 14-3-3 proteins regulate their targets, we speculate that 14-3-3 proteins could enhanced or suppressed LLPS of their binding partners (Fig. 3a) . Some 14-3-3 binding partners contain multiple [134] . a Statistical analysis of LLPS probability (p LLPS ) for 14-3-3 binding partners. According to FuzDrop, proteins with p LLPS greater than 0.61 likely form droplets spontaneously. p LLPS values for all proteins are provided in Supplementary Table S1 . b FuzDrop predicted droplet-promoting propensity profiles (p DP ) of p53, hnRNPA1, SARS-CoV-2 nucleocapsid protein, tau, and YAP. Residues with p DP > 0.60 are likely to mediate LLPS 14-3-3 binding sites which may be occupied by a 14-3-3 dimer. Upon 14-3-3 proteins binding, significant conformational changes may occur within the 14-3-3 binding partners. Therefore, LLPS of the 14-3-3 binding partners may be modulated (Mode I in Fig. 3b ). 14-3-3 proteins could also act as cross-linkers by binding to multiple partners simultaneously, thus increasing the valence of interaction for LLPS and promoting phase separation (Mode II in Fig. 3b ). PTMs are key factors regulating LLPS. 14-3-3 proteins could modulate LLPS of their binding partners by facilitating the occurrence of PTMs by recruiting enzymes (Mode III in Fig. 3b ). In the following text, we summarized YAP P46937 Transcription factor LLPS in vitro and in cells. [203, 204] the phase separation properties of five representative 14-3-3 binders and discussed how 14-3-3 proteins could play a role.

Tau is a microtubule-associated protein mainly distributed in axons. Its primary function is to regulate the assembly and spatial organization of microtubule [136, 137] . Tau also plays a role in cellular signaling and chromosome stability [138, 139] . Because hyperphosphorylation and abnormal aggregation of tau have been observed in a number of neurodegenerative diseases, tau is considered as an important target for neurodegenerative disease treatment [140] [141] [142] [143] .

The interactions between 14-3-3 and tau have been recognized for a long time and may contribute to tau aggregation [144] [145] [146] . Crystal structures and binding affinity characterization show that tau segments embracing phosphorylated residues pSer214 and pSer324 bind to the amphipathic grooves of 14-3-3 [60]. However, amphipathic groove-specific binding ligands are not able to completely inhibit binding of tau to 14-3-3 [147] . Moreover, Stefanoska et al. showed that an N-terminal 11-amino acid-long motif (residues 18-28) modulates interactions between tau and 14-3-3 [148] .

Recently, it was found that LLPS is an intrinsic property of tau and may be involved in its function and aggregation [149] [150] [151] [152] [153] . For example, tau droplets can concentrate tubulin and nucleate microtubule bundles as well as regulate the activity of microtubule-severing enzymes and the movement of molecular motors [154, 155] . LLPS also promotes tau aggregation or formation of toxic oligomers [30, 96, 97, 99, 156, 157] .

LLPS of tau is mainly driven by electrostatic interactions between the negatively charged N-terminal and the C-terminal domains and the positively charged prolinerich domain and the microtubule-binding domain [104] , whereas hydrophobic interactions are also involved [97, 105] . Many factors promoting tau aggregation have been found to promote tau phase separation [105, 153] , and several proteins, including EFhd2 [158] , PDI [159] , Hsp22 [160] , and TIA1 [157] , have been found to regulate the LLPS of tau. Considering the extensive interactions between tau and 14-3-3 proteins, we speculate that the conformational changes on tau induced by 14-3-3 may influence the phase separation of tau (mode I in Fig. 3a) . Furthermore, 14-3-3 proteins promote tau phosphorylation [161] , which may also regulate tau LLPS (mode III in Fig. 3a) .

The tumor suppressor p53 protein is a transcription factor. Its inactivation is found in many cancer types. 14-3-3 proteins regulate the transcriptional activity, stability, and cellular localization of p53 [6] . So far, three 14-3-3 binding sites located in the disordered C-terminal domain of p53 have been characterized experimentally [6] .

Early studies have demonstrated that p53 is uptaken into membrane-less cellular bodies, such as Cajal bodies and promyelocytic leukemia protein bodies [162, 163] . Droplets of p53 were first reported by Boija et at. when they mixed Fig. 3 Potential influence of 14-3-3 proteins on the LLPS propensity of 14-3-3 binding partners. a A phase diagram illustrating that LLPS of 14-3-3 binding partners may be regulated by 14-3-3 proteins. Yellow circles indicate the droplets formed. Multivalent interactions driving LLPS and recruitment of 14-3-3 into the droplets are indicated. When 14-3-3 proteins promote LLPS of their partners, the phase boundary is shifted towards lower protein concentration and lower crowder concentration. On the contrary, if 14-3-3 proteins suppress LLPS of their partners, the phase boundary is shifted towards higher protein concentration and higher crowder concentration. b Schematic illustration of modes through which 14-3-3 proteins may regulate the LLPS propensities of their targets. In mode I, a 14-3-3 dimer binds to its target and induces conformational change. In mode II, a 14-3-3 dimer binds to multiple targets, thus increasing the valence of interaction for LLPS. In mode III, a 14-3-3 dimer bridges its target with the modifying enzyme, thus facilitating the occurrence of PTMs p53 with transcriptional coactivator MED1 [35] . Later, anomalous liquid condensates of p53 were observed by Safari et al. when they explored the aggregation process of p53 at near-physiological conditions and in crowded environments [164] . p53 also forms droplets when overexpressed in yeast [165] . While experiments using recombinant p53 suggest that electrostatic interactions between the negatively charged N-terminal domain and the positively charged C-terminal domain play a key role in mediating p53 droplet formation [166] , some mutations are found to markedly promote p53 condensation in cancer cells [167, 168] . Though the function of p53 condensates remains elusive [169] , formation of p53 droplets may be related to p53 aggregation [164, 165, 168, 170] .

Oligomerization is a key factor in LLPS as it generates multi valences and enhances intermolecular interactions [109, 132, 171] . Interactions with 14-3-3 proteins are found to enhance the tetramerization of p53 [172] . In this context, 14-3-3 proteins are expected to enhance the LLPS propensity of p53 and have an effect on the subsequent functioning process (mode II in Fig. 3a ).

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that regulate diverse biological processes. hnRNPA1 is concentrated in the nucleus and involved in mRNA processing. During stress, hnRNPA1 is sequestered in the cytoplasm and forms stress granules with other RNA-binding proteins and mRNA [173] . A 14-3-3 binding motif is located on the N-terminus, where phosphorylation on Ser4/6 is required for hnRNPA1/ 14-3-3 interactions [174] . 14-3-3 can mediate re-entry of hnRNPA1 into the nucleus or cooperate with hnRNPA1 to control splicing of genes under stress conditions [174, 175] . hnRNPA1 consists of two folded RNA recognition motifs and a low complexity domain. While LLPS of hnRNPA1 is mainly driven by the low complexity domain via aromatic-aromatic and aromatic-arginine interactions, the folded RNA recognition motifs modify the phase behavior through intramolecular electrostatic interactions between the low complexity domain and the RNA recognition motifs [32, 176] . LLPS of hnRNPA1 has been suggested to contribute to the assembly of stress granules and the formation of amyloid-like fibers [32, 177, 178] . Since conditions that favor LLPS also enhance hnRNPA1 aggregation, it is suggested that LLPS is on pathway to its aggregation [179] .

14-3-3 proteins have been shown to regulate the component of stress granules. MK2-induced phosphorylation of tristetraprolin at Ser52 and Ser178 promotes formation of tristetraprolin/14-3-3 complexes, resulting in exclusion of tristetraprolin from arsenite-induced stress granules [180] . Moreover, 14-3-3 proteins were able to regulate the distribution of hMex-3B in P-bodies and stress granules [181] . Importantly, both tristetraprolin and hMex-3B are predicted to undergo phase separation spontaneous with p LLPS values of 0.9986 and 0.9988, respectively. Modulating the component of stress granules by 14-3-3 or building up physical cross-linking between hnRNPA1 may influence the material properties or functions of stress granules (mode II in Fig. 3a ).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the worldwide pandemic of the coronavirus disease 2019 (COVID-19) [182] . The SARS-CoV-2 genome encodes about 30 proteins, among which the nucleocapsid (N) protein is the most abundant viral protein in infected cells [183] [184] [185] . N protein is a multifunctional protein. It packages viral RNA into virions and plays a critical role in virus transcription and assembly [186] .

14-3-3 proteins have been reported to regulate the shuttling of SARS-CoV N protein between the nucleus and the cytoplasm [187] . Recently, Tugaeva et al. characterized the interactions between N protein and 14-3-3 proteins [188] . They found that N protein interacts with all seven human 14-3-3 isoforms with K d values in low micromolar range. The main binding site for N protein to bind 14-3-3 is phosphorylated Ser197, which is located in the central IDR.

The phase separation propensity of N protein has been subjected to extensive studies. N protein undergoes LLPS with RNA where the N-terminal RNA-binding domain, the central IDR, and C-terminal dimerization domain play an essential role [39, [189] [190] [191] [192] [193] [194] . LLPS of N protein is modulated by phosphorylation at the serine/arginine-rich region by CDK1, GSK-3β, or SRPK1 [38, 193, 195] . The N protein/RNA condensates recruit RNA polymerase complex of SARS-CoV-2, which may provide a mechanism for efficient transcription of viral RNA [38] . N protein also phase separates with G3BP or hnRNPA2 into stress granules [190, 193, 196, 197] . Furthermore, LLPS of N protein may be associated with the dysfunctional inflammatory responses and antiviral immunity [194, 198] . Consequently, modulating the phase separation propensity of N protein could be a potential treatment for COVID-19 [39, 199] .

Though the influence of 14-3-3 on LLPS of N protein has not been investigated so far, binding of 14-3-3 to N protein is expected to modify the conformation of the central IDR or introduce intermolecular crossing-linking, which could play a role in modulating the phase separation propensity of N protein (mode I and mode II in Fig. 3a) . Recruitment of 14-3-3 into N protein condensates may also regulate the signaling pathways involved in immune response.

The Yes-associated protein (YAP) is a key transcriptional coactivator of the Hippo pathway [200] . Unphosphorylated YAP is located in the nucleus and interacts with the TEAD transcription factors [201] . Upon association with TEAD, YAP induces expression of genes involved in anti-apoptotic processes [202] . On the contrary, when YAP is phosphorylated by LATS1-2, it binds to 14-3-3 proteins and remains in the cytosol [202] . Therefore, 14-3-3 proteins play a role in regulating the Hippo pathway.

To date, there are two studies showing that YAP undergoes phase separation. In one study, Yu et al. found that interferon-γ promotes phase separation of YAP after anti-PD-1 therapy in tumor cells [203] . YAP droplets induced by interferon-γ are localized in the nucleus and recruit TAZ, TEAD4, EP300, and MED1, forming a transcriptional hub for gene expression. Therefore, disrupting LLPS of YAP suppresses tumor cell growth [203] . In the other study, Cai et al. investigated the phase separation behavior of YAP under hyperosmotic stress [204] . In hyperosmotically stressed cells, YAP condensates are observed in both cytoplasm and nucleus. Importantly, the components of cytoplasmic and nuclear YAP condensates are different. Cytoplasmic YAP condensates concentrate kinase, including LATS1 and NLK, whereas nuclear YAP condensates contain transcription factor and transcription regulator, such as TEAD1 and TAZ. While both studies confirm the essential role of C-terminal IDR of YAP in LLPS, formation of YAP condensates is regulated by Ser127 and Ser128 phosphorylation [204] . The Ser127 phosphorylation creates a binding site for 14-3-3 proteins and promotes formation of cytoplasmic YAP condensates, whereas phosphorylation on Ser128 abolishes YAP/14-3-3 binding and suppresses LLPS of YAP in the cytoplasm [204, 205] . Therefore, 14-3-3 proteins may play a critical role in regulating the formation of cytoplasmic YAP condensates which sequester YAP from the nucleus (mode I and II in Fig. 3a ).

In the past ten years, our understanding on LLPS has achieved rapid development. Increasing studies show that LLPS plays a fundamental role in formation of membraneless organelles and cellular condensates, and dysregulation of LLPS is closely related to human diseases. 14-3-3 proteins interact with various targets and form an interacting hub for many cellular signaling pathways. While numerous investigations have been performed on 14-3-3 proteins, its involvement in LLPS is overlooked. To reveal the potential correlation between 14-3-3 proteins and LLPS, in this review, we summarized the LLPS propensity of 14-3-3 binding partners and found that about one half of them may undergo LLPS spontaneously. We further analyzed the phase separation behavior of representative 14-3-3 binders and discussed how 14-3-3 protein may be involved. By modulating the conformation and valence of interactions and recruiting other molecules, we speculate that 14-3-3 proteins can efficiently regulate the functions of their targets in the context of LLPS. Considering the critical roles of 14-3-3 proteins, there is an urgent need for investigating the involvement of 14-3-3 proteins in the phase separation process of their targets and the underlying mechanisms.

All data are included in the article and its supplementary information files. (1995). 14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF. 

Physiological and biochemical aspects of nervous integration

Structural basis of 14-3-3 protein functions

The 14-3-3 proteins as important allosteric regulators of protein kinases

14-3-3 proteins: a historic overview

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

Visualization and biochemical analyses of the emerging mammalian 14-3-3-phosphoproteome

Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

Human 14-3-3 paralogs differences uncovered by cross-talk of phosphorylation and lysine acetylation

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation

Bcr and Raf form a complex in vivo via 14-3-3 proteins

An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation

Role of a pineal cAMP-operated arylalkylamine Nacetyltransferase/14-3-3-binding switch in melatonin synthesis

Structural insight into the 14-3-3 protein-dependent inhibition of protein kinase ASK1 (Apoptosis Signal-regulating kinase 1)

The expression of seven 14-3-3 isoforms in human meningioma

Isoform-specific expression of 14-3-3 proteins in human astrocytoma

PTPIP51, a positive modulator of the MAPK/Erk pathway, is upregulated in glioblastoma and interacts with 14-3-3beta and PTP1B in situ

Prognostic significance of 14-3-3gamma overexpression in advanced non-small cell lung cancer

Isoformspecific expression of 14-3-3 proteins in human lung cancer tissues

Breast cancer cell motility is promoted by 14-3-3gamma

Characterization of the expression and prognostic value of 14-3-3 isoforms in breast cancer

Overexpression of 14-3-3epsilon predicts tumour metastasis and poor survival in hepatocellular carcinoma

14-3-3epsilon overexpression contributes to epithelial-mesenchymal transition of hepatocellular carcinoma

COPS5 and LASP1 synergistically interact to downregulate 14-3-3sigma expression and promote colorectal cancer progression via activating PI3K/AKT pathway

14-3-3 binds to and mediates phosphorylation of microtubule-associated tau protein by Ser9-phosphorylated glycogen synthase kinase 3beta in the brain

Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease

Specification of 14-3-3 proteins in Lewy bodies

14-3-3 proteins in Lewy bodies in Parkinson disease and diffuse Lewy body disease brains

14-3-3 proteins and zeta isoform containing neurofibrillary tangles in patients with Alzheimer's disease

Examination of stress-related genes in human temporal versus occipital cortex in the course of neurodegeneration: involvement of 14-3-3 zeta in this dynamic process

14-3-3 proteins: novel pharmacological targets in neurodegenerative diseases

Emerging roles of 14-3-3gamma in the brain disorder

14-3-3 proteins in neurological disorders

Liquid-liquid phase separation induces pathogenic tau conformations in vitro

Tau protein liquid-liquid phase separation can initiate tau aggregation

Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles

Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein tau

Phase transitions in the assembly of multivalent signalling proteins

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife, 5, e13571

Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity

A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins

Liquid-liquid phase separation of tau protein: the crucial role of electrostatic interactions

Electrostatically driven complex coacervation and amyloid aggregation of tau are independent processes with overlapping conditions

Physical principles underlying the complex biology of intracellular phase transitions

The role of post-translational modifications in the phase transitions of intrinsically disordered proteins

A guide to regulation of the formation of biomolecular condensates

Liquid droplet formation by HP1alpha suggests a role for phase separation in heterochromatin

Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity

Phosphoregulated FMRP phase separation models activity-dependent translation through bidirectional control of mRNA granule formation

Phase separation of zonula occludens proteins drives formation of tight junctions

LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force

Pi-Pi contacts are an overlooked protein feature relevant to phase separation

FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-pi interactions

Mechanistic view of hnRNPA2 low-complexity domain structure, interactions, and phase separation altered by mutation and arginine methylation

N-terminal acetylation modestly enhances phase separation and reduces aggregation of the low-complexity domain of RNA-binding protein fused in sarcoma

Acetylation of intrinsically disordered regions regulates phase separation

Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation

Ubiquitination of G3BP1 mediates Cell Biochemistry and Biophysics stress granule disassembly in a context-specific manner

Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

Poly(ADP-ribose): a dynamic trigger for biomolecular condensate formation

Compositional control of phase-separated cellular bodies

Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1

Dynamics of component exchange at PML nuclear bodies

Insight into membraneless organelles and their associated proteins: drivers, clients and regulators

Assembly of mitotic structures through phase separation

Phase separation of a yeast prion protein promotes cellular fitness

Stresstriggered phase separation is an adaptive, evolutionarily tuned response

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing

Protein phase separation and its role in tumorigenesis

ALS mutations disrupt phase separation mediated by alphahelical structure in the TDP-43 low-complexity C-terminal domain

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics

Widespread occurrence of the droplet state of proteins in the human proteome

The MIntAct project-IntAct as a common curation platform for 11 molecular interaction databases

Roles of tau protein in health and disease

Tau regulates the localization and function of End-binding proteins 1 and 3 in developing neuronal cells

The many faces of tau

Atypical, non-standard functions of the microtubule associated tau protein

Tau-based therapies in neurodegeneration: opportunities and challenges

An overview on the clinical development of tau-based therapeutics

Alzheimer disease: an update on pathobiology and treatment strategies

Tau proteins and tauopathies in Alzheimer's disease

14-3-3/Tau Interaction and Tau Amyloidogenesis

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

Interaction of 14-3-3 zeta with microtubule-associated protein tau within Alzheimer's disease neurofibrillary tangles

Inhibition of 14-3-3/tau by hybrid small-molecule peptides operating via two different binding modes

An N-terminal motif unique to primate tau enables differential protein-protein interactions

Liquid-liquid phase separation of Tau by self and complex coacervation

Liquid-liquid phase separation of tau: From molecular biophysics to physiology and disease

Liquid-liquid phase separation of tau protein in neurobiology and pathology

Tau condensates

The structure and phase of tau: from monomer to amyloid filament

Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase

Microtubules gate tau condensation to spatially regulate microtubule functions

Liquid-liquid phase separation of tau driven by hydrophobic interaction facilitates fibrillization of tau

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

EFhd2 affects tau liquid-liquid phase separation

Phase separation and cytotoxicity of tau are modulated by protein disulfide isomerase and s-nitrosylation of this molecular chaperone

Small heat shock protein 22 kDa can modulate the aggregation and liquid-liquid phase separation behavior of tau

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubuleassociated tau phosphorylation complex

Cajal bodies: a long history of discovery

The function of PML in p53-dependent apoptosis

Anomalous dense liquid condensates host the nucleation of tumor suppressor p53 fibrils. iScience

Tumor suppressor protein p53 expressed in yeast can remain diffuse, form a prion, or form unstable liquid-like droplets. iScience, 24, 102000

Liquidlike droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains

Identification of small molecules that modulate mutant p53 condensation. iScience

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands

p53 mediates target gene association with nuclear speckles for amplified RNA expression

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils

Phase-separated condensate-aided enrichment of biomolecular interactions for high-throughput drug screening in test tubes

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers

Heterogeneous nuclear ribonucleoproteins: implications in neurological diseases

hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling

hnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the alternative splicing response to oxaliplatin-mediated DNA damage

Interplay of folded domains and the disordered lowcomplexity domain in mediating hnRNPA1 phase separation

Formation and maturation of phase-separated liquid droplets by RNA-binding proteins

Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets

Electrostatic modulation of hnRNPA1 low-complexity domain liquid-liquid phase separation and aggregation

MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay

Interaction with 14-3-3 adaptors regulates the sorting of hMex-3B RNA-binding protein to distinct classes of RNA granules

A new coronavirus associated with human respiratory disease in China

Proteomics of SARS-CoV-2-infected host cells reveals therapy targets

The global phosphorylation landscape of SARS-CoV-2 infection

The total number and mass of SARS-CoV-2 virions

The coronavirus nucleocapsid is a multifunctional protein

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Liquid-liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA

SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs

Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid

Characterization of SARS-CoV-2 nucleocapsid protein reveals multiple functional consequences of the C-terminal domain. iScience, 24, 102681

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Targeting liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein promotes innate antiviral immunity by elevating MAVS activity

Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical basis for its dual functions

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation into stress granules through its N-terminal intrinsically disordered region

SARS-CoV-2 nucleocapsid protein phase separates with G3BPs to disassemble stress granules and facilitate viral production

RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-kappaB hyperactivation and inflammation

Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2

An updated understanding of the role of YAP in driving oncogenic responses

The biology of YAP/TAZ: hippo signaling and beyond

Mechanisms of Hippo pathway regulation

Interferon-gamma induces tumor resistance to anti-PD-1 immunotherapy by promoting YAP phase separation

Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression

Osmotic stress-induced phosphorylation by NLK at Ser128 activates YAP

Phase separation of Axin organizes the beta-catenin destruction complex

Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient beta-catenin degradation

Phase separation of the Cep63-Cep152 complex underlies the formation of dynamic supramolecular self-assemblies at human centrosomes

Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies

TGF-betainduced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

Atomic-level insight into mRNA processing bodies by combining solid and solution-state NMR spectroscopy

A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping

Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation

In vitro reconstitution of a cellular phase-transition process that involves the mRNA decapping machinery

The ERC1 scaffold protein implicated in cell motility drives the assembly of a liquid phase

GIT/PIX condensates are modular and ideal for distinct compartmentalized cell signaling

Site-specific ubiquitylation acts as a regulator of linker histone H1

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

The SH3 domain of Fyn kinase interacts with and induces liquid-liquid phase separation of the low-complexity domain of hnRNPA2

Weak binding to the A2RE RNA rigidifies hnRNPA2 RRMs and reduces liquid-liquid phase separation and aggregation

Regulatory expansion in mammals of multivalent hnRNP assemblies that globally control alternative splicing

Inhibition of HSF1 and SAFB granule formation enhances apoptosis induced by heat stress

HSF1 phase transition mediates stress adaptation and cell fate decisions

The huntingtin inclusion is a dynamic phase-separated compartment

A phosphatidic acid-binding lncRNA SNHG9 facilitates LATS1 liquid-liquid phase separation to promote oncogenic YAP signaling

The F-actin-binding MPRIP forms phase-separated condensates and associates with PI(4,5)P2 and active RNA polymerase II in the cell nucleus

Stress-induced nuclear condensation of NELF drives transcriptional downregulation

C9orf72 poly(PR) dipeptide repeats disturb biomolecular phase separation and disrupt nucleolar function

Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation

Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

Condensation of pericentrin proteins in human cells illuminates Cell Biochemistry and Biophysics phase separation in centrosome assembly

Phase separation of Polo-like kinase 4 by autoactivation and clustering drives centriole biogenesis

Acto-myosin force organization modulates centriole separation and PLK4 recruitment to ensure centriole fidelity

DNA repair by Rad52 liquid droplets

Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52. Genes and Development

ATP biphasically modulates LLPS of SARS-CoV-2 nucleocapsid protein and specifically binds its RNA-binding domain

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

SON and SRRM2 are essential for nuclear speckle formation

Pressure sensitivity of SynGAP/PSD-95 condensates as a model for postsynaptic densities and its biophysical and neurological ramifications

SynGAP isoforms differentially regulate synaptic plasticity and dendritic development

RNA stores tau reversibly in complex coacervates

Acetylation disfavors tau phase separation

Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions. Elife, 8, e42571

Lysine/RNA-interactions drive and regulate biomolecular condensation

The acetyl mimicking mutation, K274Q in tau, enhances the metal binding affinity of tau and reduces the ability of tau to protect DNA

Zinc promotes liquid-liquid phase separation of tau protein

The proline-rich domain promotes Tau liquid-liquid phase separation in cells

Interplay between tau and alpha-synuclein liquid-liquid phase separation

The positive regulation loop between NRF1 and NONO-TFE3 fusion promotes phase separation and aggregation of NONO-TFE3 in NONO-TFE3 tRCC

Inositol polyphosphate multikinase inhibits liquid-liquid phase separation of TFEB to negatively regulate autophagy activity

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins

USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis